CueO is a protein that plays a role in protecting enzymes in periplasm of Escherichia coli from copper-mediated damage. CueO produced from Escherichia coli is a monomer with 516 residues and a molecular weight of 56555 Da, while N-terminal 28 residues serve as a signal sequence and are cleaved upon transferring into periplasm so as to give a mature protein with 488 residues and a molecular weight of 53409 Da.
CueO includes 4 copper atoms per molecule, and it is classified as a multi-copper oxidase family. CueO is different from laccase as it has significantly low activity for oxidizing a phenol compound such as 2,6-dimethoxyphenol or catechol, or does not oxidize at all. CueO has a binding site for 5th copper, and in the presence of exogenous copper, it exhibits a phenol oxidase activity. CueO is a protein encoded by a gene known as yacK, and amino acid sequences thereof and DNA sequences encoding thereof are reported in Non-Patent Document 1. The fundamental characteristics are reported in Non-Patent Document 2 and Non-Patent Document 3.
Non-Patent Document 2 is disclosing that CueO exhibits significantly low enzymatic activity for oxidizing p-phenylenediamine, 2,6-dimethoxyphenol, or the like, or does not exhibit the activity at all, but this enzymatic activity can be improved under co-existence of divalent copper. However, the above-mentioned enzymatic activity of CueO in the copper-free system is not sufficient for a use, particularly, in dyeing.
From the past, for dyeing keratin fiber including hair and various fibers, oxidation dyes have been used to maintain the dyeing effect. The oxidation dye achieves dyeing by color development via an oxidation reaction and a polymerization reaction. The oxidation dye is for forming a permanent dyeing agent.
In order to proceed the oxidation and polymerization reaction of the oxidation dye, an oxidizing agent such as hydrogen peroxide has been used from the past. In such case, when the oxidation dye penetrated into the hair undergoes oxidative polymerization, high molecular weight coloring materials are formed and deposited inside the hair cortex. Here, since the hydrogen peroxide is highly reactive, full attention is required in handling. In addition, damages to scalp or hair due to a combination use of alkali and hydrogen peroxide have been the problem. Further, a using method thereof is complicated as the dye is a two-part type agent in which an oxidation dye and an alkaline agent are blended as a first agent and hydrogen peroxide is blended as a second agent which are mixed at the time of use.
In consequent, there has been made an attempt to carry out a direct oxidative polymerization of oxidation dye by employing an enzyme instead of an oxidizing agent. For such enzyme, multi-copper oxidase has been suggested. The multi-copper oxidase directly oxidizes the substrate without causing reactive oxygen species such as hydrogen peroxide, and thus is suited for various chemical reactions such as color development, discoloring, and decomposition, which are caused by extremely stable oxidation of the substrate.
As such multi-copper oxidase for a use in dyeing, specifically, a culture from a strain belonging to the genus Flammulina, wherein the culture has phenol oxidase-like activity (see Patent Document 1); a neutral phenol oxidase derived from a basidiomycete belonging to the genus Flammulina (see Patent Document 2); or a laccase derived from Myceliophthora thermophila (see Patent Document 3) has been proposed for a use.
When dyeing of keratin fiber is carried out under an acidic or neutral condition, discoloring is more easily achieved. For the purpose of improving and maintaining the dyeing effect by allowing swelling of keratin fiber such for the oxidation dye to penetrate into the hair cortex and thereby allowing deposition of the high molecular weight dye materials inside the hair cortex, dyeing with the oxidation dye is preferably carried out under an alkaline condition.
However, neutral phenol oxidases disclosed in Patent Document 1 and Patent Document 2 have an optimum pH near a neutral pH, and thus are not suited for a use in an alkaline condition. Also, the oxidases have been difficult to be used in practical because the stability near a neutral pH which is the optimum pH is low. Further, there has been a problem in these neutral phenol oxidases that color matching with a direct dye is difficult as they have a property of catalyzing the decomposition/discoloring of a direct dye.
The laccase derived from Myceliophthora thermophila disclosed in Patent Document 3 also has an optimum pH of 6.5, thus is not suited for a use in an alkaline condition.
Patent Document 1: Pamphlet of Int. Pub. No. 2004/020617
Patent Document 2: Pamphlet of Int. Pub. No. 2004/078958
Patent Document 3: PCT Japanese Translation Patent Publication No. 10-501137
Non-Patent Document 1: ‘SCIENCE’, 1997, 227(5331), 1453-1474
Non-Patent Document 2: ‘JOURNAL OF BACTERIOLOGY’, August 2001, 4866-4875
Non-Patent Document 3: ‘THE JOURNAL OF BIOLOGICAL CHEMISRY’, August 2003, Vol. 278, 34, 31958-31963